Over the past few years, extreme summer temperatures have caused an alarming increase in deaths due to hyperthermia. Elevated body temperatures lead to seizures, respiratory distress, brain damage and death (Glazer, 2005). The time between hyperthermic neuronal failure and cell death and permanent thermal damage is brief (Tryba and Ramirez, 2004). Because neural output failure occurs before permanent thermal damage (Tryba and Ramirez, 2004), this narrow window of time is available for the protection and recovery of neural circuit function until return to normal temperatures. Fast neural recovery allows the hyperthermic individual to resume normal pulmonary and cardiac functioning.
As will be outlined further below, it is known that the cGMP-dependent protein kinase G (PKG) pathway may modulate neural potassium (K+) channel activity, and that K+ channel activity may be involved in neural thermoprotection. The involvement of Nitric Oxide (NO) in heatstroke has also been demonstrated, although the nature of this involvement has not been clarified. However, due to the multiplicity of other enzyme pathways which modulate K+ activity and the multiplicity of other channels and other systems which modulate neural thermal response, any relationship between the PKG pathway, K+ channel activity and NO has not been clearly demonstrated.
The PKG enzyme pathway is known, and an outline of the PKG pathway as it is involved in ion channel regulation is provided to assist in understanding:
Briefly, nitric oxide (NO) is produced by various NO synthases (NOS), some of which are activated by a rise in intracellular Ca2+. Many NO effects are mediated through direct activation of the soluble guanylyl cyclase (sGC), an enzyme generating the second messenger cyclic guanosine-3′,5′-mono-phosphate (cGMP). sGC is stimulated by NO to catalyze the formation of cGMP. cGMP is a cyclic nucleotide second messenger with effects on many pathways, one of which is the cGMP-dependent protein kinase (PKG) enzyme pathway. PKG is an enzyme that transfers a phosphate group from ATP to an intracellular protein, increasing or decreasing its activity.
Both the DNA sequence and protein function of PKG are conserved across the animal kingdom including mammalians. PKG genes have been isolated from various animals spanning a wide variety of taxa ranging from humans (Sandberg et al., 1989) to even the malaria-causing protozoans Plasmodium falciparum (Gurnett et al., 2002). The protein phylogenetic analysis using 32 PKG sequences that include 19 species has shown the highly conserved link between PKG and behaviour in fruit flies, honey bees and nematodes (Fitzpatrick et al., 2004).
It is known that PKG may modulate neural ion channel activity i.e. neural potassium (K+) activity (Renger, 1999). A variety of mechanisms for this effect have been suggested, ranging from direct phosphorylation of the K+ channel by PKG, to the opposing indirect dephosphorylation of the K+ and other channels by phosphatases such as PP2A, which are themselves activated by PKG phosphorylation (Schiffmann et al., 1998; White et al., 1993; White, 1999; Zhou et al., 1996; Zhou et al., 1998). It has been suggested on theoretical grounds that future work in the area of regulation of the PKG pathway might yield some neuroprotective effects but no supporting evidence was provided (Jayakar and Dikshit, 2004).
Effects of cGMP inhibitors on neural ion channels were proposed to lead to pain relief compounds (Liu et al., 2004).
Most research on ion channel direct phosphorylation focuses on the enzyme PKA, which is known to modulate many ion channels (Wang et al., 1999; Zeng et al., 2004). PKA often functions in opposition to PKG, but may function in the same direction.
There is extensive literature related to PKG's known roles in the regulation of smooth muscle and other non-neural tissues, where it may modulate ion channels. A significant amount of medical and pharmaceutical publications relate to such drugs as sildenafil (Viagra) and nitroglycerine for treatment of penile dysfunction or angina (Corbin and Francis, 1999; Patel and Diamond, 1997).
U.S. Pat. No. 6,300,327 to Knusel et al. teaches compositions and methods for use in modulating neurotrophin activity. Neurotrophin activity is modulated by administration of an effective amount of at least one compound which potentiates neurotrophin activity. This patent specifically teaches the potentiation of NT-3 by KT5823, and suggests that this potentiation provides a model for therapeutic intervention in a variety of neuropathological conditions.
U.S. Pat. No. 6,451,837 to Baskys teaches inhibition of the mitogen-activated protein kinase (MAPK) cascade that can lead to nerve cell death. In this context, Okadaic acid, an inhibitor of protein phosphatase was found to increase nerve cell death.
U.S. Pat. No. 6,476,007 to Tao et al. investigated the role of the NO/cGMP signalling pathway in spinal cord pain.
It is known that K+ channel activity may be involved in neural thermoprotection. As canvassed further below, existing literature has considered what happens to neural function during heat-induced malfunction; mechanisms known to provide heat tolerance, such as induction of the heat shock genes and proteins; and investigations into a direct mechanism for neural thermoprotection by K+ channel modulation.
Abnormal K+ concentrations have been associated with various neural failure scenarios such as spreading depression, ischemia, diabetic coma, and hyperthermia (Somjen, 2001; Somjen, 2002).
Recent research by one of the inventors suggests that prior stress down-regulates neuronal K+ currents leading to thermal protection (Robertson, 2004). This was based on findings that prior heat shock provides protective effects against future heat shock and that neuronal K+ currents are effected by prior heat stress (Ramirez et al., 1999).
Other investigations have suggested that K+ channels are involved in neural excitability in response to other stresses such as reactive oxygen (Wang et al., 2000).
It is known that Nitric Oxide (NO) is involved in heatstroke. NO is a pervasive signaling mechanism in the body. One of its functions is to stimulate the PKG pathway, but this is only one of the many routes by which it acts. For instance, NO may be directly toxic to cells, or it may modulate cGMP-dependent ion channels without the involvement of PKG. Thus, prior art showing a correlation of heatstroke with brain NO production does not establish the involvement of the PKG pathway.
For example, NO was shown to be elevated in heatstroke rats (Canini et al., 1997) but a mechanism of involvement in heatstroke was not demonstrated. More recently, it was shown that prior treatments such as Naltrexone, an opiate inhibitor, could reduce NO production during heatstroke, with some beneficial effects such as reduction of body temperature (Sachidhanandam et al., 2002). However, neural protection was not identified as a possible application. Nor was the treatment rapid or responsive to an existing condition: it required administration before heat application.
U.S Pat. No. 6,511,800 to Singh teaches methods for the inhibition of inducible nitric oxide synthesis and the production of NO, and treating of nitric oxide or cytokine mediated disorders.
U.S. Pat. No. 5,990,177 to Brown teaches methods and compositions for stimulating cellular NO synthesis, cGMP levels and PKG activity for the purposes of treating diseases mediated by deficiencies in the NO/cGMP/PKG pathway. Brown does not implicate this system in the treatment of hyperthermic conditions.
In another study, an inhibitor (L-NNA) of NO synthesis administered prior to heat exposure provided small protective effects against febrile convulsions, which the authors attributed to reduction of the directly neurotoxic effects of NO (Klyueva et al., 2001). Again, this is an example of prior treatment, modest protective effects, and no implication of the PKG pathway. Klyueva et al. point out that published data on the role of NO in the pathogenesis of seizures are contradictory: both the anti- and proconvulsant effect of NO have been reported.
Indeed, another study of inhibitors of NO synthesis showed that these reduced heat tolerance (Canini et al., 2001). Another study concluded that NO did not have a pathogenic role in heatstroke at doses given in the study (Gulec and Noyan, 2001). More recently, the inhibitor of NO production aminoguanadine was shown to have some protective effects against heatstroke-induced intracranial hypertension and cerebral ischemic injury by inhibition of cerebral iNOS-dependent NO (Chang et al., 2004).
It has further been observed that NO and cGMP may be involved in thermosensing and thermoregulation, i.e. control of body temperature (Gerstberger, 1999).
There are known treatments for hyperthermia. For example, U.S. Pat. No. 6,846,845 to Takahashi et al. teaches a heat shock protein inducer, which induces heat shock protein in the heart, for preventing or treating ischemic disease or ischemia/reperfusion injury. As pointed out in the patent, heat shock proteins are a family of endogenous protective proteins generated in response to stress, including hyperthermia.
Hyperthermia is currently treated by chilling the person suffering from hyperthermia. This treatment may be insufficient or too late, however, to trigger the neurological recovery necessary to keep the basic systems of the body functioning.
In nature, heat shock protein synthesis protects organisms from sustained high heat conditions over a matter of hours and days. There are currently no therapeutic compounds to prevent hyperthermia, particularly in the short term.
The primary therapy of heatstroke is cooling the patient. The traditional cooling methods such as ice water soaks, immersion in ice baths, and use of cooling blankets as well as application of ice to the groin, neck, and axilla have been used as cooling modalities in heatstroke. Cooling heatstroke patients using these modalities invariably leads to shivering. Diazepam is the drug of choice to decrease shivering and at a dose of 5-10 mg intravenously, abolishes shivering reflexes during cooling. Chlorpromazine (10-25 mg administered slowly IV) also has been recommended to reduce shivering during cooling. Unfortunately, because chlorpromazine may cause hypotension and arrhythmias, and decreases the seizure threshold, it is considered a second-line agent (after diazepam) for controlling shivering associated with cooling.
Dantrolene is the treatment of choice for malignant hyperthermia and has been proposed as a treatment for heatstroke (administered at 1 mg/kg). The mechanism of action appears to involve inhibition of calcium release in skeletal muscle. The major side effects are muscle weakness and nausea. Although effective, studies have shown that there was no significant difference in the number of hospital days in the control and dantrolene treated groups, and mortality and morbidity rates were unchanged. The overall prognosis of heatstroke depends on how fast the cooling therapy is applied to the patients.
Treatment of febrile seizures traditionally consists of continuous or intermittent therapy with anticonvulsants or no therapy. Anticonvulsants have not been proved to prevent subsequent development of febrile seizures nor is there any evidence that febrile seizures cause structural or cognitive damage. Carbamazepine and phenytoin have not been shown to prevent recurrence, whereas phenobarbital reduced the incidence of febrile seizures from 25 per 100 children to five per 100 children. However, hyperactivity and hypersensitivity reactions can occur with this drug. Valproic acid is considered as effective as phenobarbital in preventing recurrence but is associated with several potentially serious adverse effects, such as thrombocytopenia, weight changes and fatal hepatotoxicity. Intermittent treatment may include diazepam, which is associated with a 44 percent reduction in risk per patient year. However, its sedative effects can obscure signs and symptoms of a developing central nervous system infection. Antipyretics have also been used to prevent recurrence, but studies show that they do not have this effect.